Sample collection system with caspase inhibitor

ABSTRACT

A collection container and a method for collecting a biological sample, particularly whole blood, includes at least one stabilizing agent in an amount effective to inhibit apoptosis. The stabilizing agent comprises or consists of one or more caspase inhibitors.

FIELD OF THE INVENTION

The present invention is directed to a method and device for collectingand stabilizing a biological sample, directly from a patient and/orcultured cells, either from animals or humans.

BACKGROUND OF THE INVENTION

In clinical diagnostic and clinical research settings, it has often beennecessary to collect biological samples such a whole blood, red bloodcell concentrates, platelet concentrates, leukocyte concentrates,tissue, bone marrow apirates, cerebral spinal fluid, feces, urine,saliva, oral secretions, nasal secretions and the like in variouscontainers or tubes for subsequent analysis or in vitro culture.Typically, the samples must then be transported to a different location,such as a laboratory, where personnel conduct specific tests on ormanipulate the samples for future testing or implantation.

Generally, a considerable amount of time elapses between obtaining thesample and analyzing or manipulating it. A common and recurring problem,therefore, is the maintenance of the biological sample in a manner thatprevents degradation, alteration or destruction of essential materialsduring the manipulations and/or preparations preceding analysis orimplantation of the biological sample as a test specimen.

SUMMARY OF THE INVENTION

All cells and tissues undergo a process of apoptosis, or programmed celldeath (Kerr, Wyllie, and Currie, 1972 Brit J. Cancer 26:239) as anatural process in development and morphogenesis to remove unwanted oraged cells. Cells undergoing apoptosis are morphologically andbiochemically distinguishable from cells involved in necrosis, which isassociated with acute injury to cells. Apoptosis is characterized bynuclear chromain condensation, DNA fragmentation, cell membranealterations, caspase activity, cytoplasmic shrinking, dilatedendoplasmic reticulum, and membrane blebbing.

Apoptotic death can be triggered by a wide variety of stimuli, and notall cells necessarily will die in response to the same stimulus. Amongthe more studied death stimuli is DNA damage by irradiation or drugsused for cancer chemotherapy. Some hormones such as corticosteroids leadto death in particular cells (e.g., thymocytes), although other celltypes may be stimulated. Apoptosis is also evident in blood samplescollected by phlebotomy and tissue samples.

Biochemical correlates of these morphological features have emergedduring the subsequent years of study of this phenomenon. The first andmost dramatic is DNA fragmentation, which was described by Wyllie in1980. When DNA from apoptotically dying cells was subjected to agarosegel electrophoresis, ladders with ˜200 bp repeats were observed,corresponding histone protection in the nucleosomes of native chromatin.Subsequent pulsed field gel techniques have revealed earlier DNAcleavage patterns into larger fragments. Since even a few doublestranded DNA breaks will render the cell unable to undergo mitosissuccessfully, such DNA fragmentation can be regarded as a biochemicaldefinition of death.

Changes in the cell membrane in the apoptotic cell trigger phagocytosisby non-activated macrophages. Apoptotic cells lose the normalphospholipid asymmetry in their plasma membrane, as manifested by theexposure of normally inward-facing phosphatidyl serine on the externalface of the bilayer. Macrophages can recognize this exposed lipidheadgroup via an unknown receptor, triggering phagocytosis andelimination of these damaged cells from the organism.

Apoptosis in granulocytes present in whole blood samples post phlebotomyis manifested by loss of key features of this important cell type. CD16b(a granulocyte cell surface marker), oxidative burst function, membranelipid polarity and hypodiploidy all decline, degrading the quality ofthe sample for analysis. Consequently, aberrant or inaccurate analyticaltest results which depend on these cell paramaters can occur with wholeblood samples analyzed after 8 hours from the time of phlebotomy.

Another biochemical hallmark of apoptotic death is the activation ofcaspases, which are cysteine proteases related to ced-3, the “deathgene” of the nematode Caenorhabditis elegans. Caspases seem to be widelyexpressed in an inactive proenzyme form in most cells. Their proteolyticactivity is characterized by their unusual ability to cleave proteins ataspartic acid residues, although different caspases have different finespecificities involving recognition of neighboring amino acids. Activecaspases can often activate other pro-caspases, allowing initiation of aprotease cascade. Persuasive evidence that these proteases are involvedin most examples of apoptotic cell death has come from the ability ofspecific caspase inhibitors to block cell death, as well as thedemonstration that knockout mice lacking caspase 3, 8 and 9 fail tocomplete normal embryonic development.

In the area of blood collection, a common additive generally used inblood samples prior to centrifuging to separate the blood into celllayers is an anticoagulation additive. Typically, the anticoagulationadditive is a potassium or sodium salt of ethylene diamine tetraaceticacid (EDTA), a buffered citrate, or heparin in an aqueous solution orcrystalline coating of the interior of the collection vessel. Bloodcollection tubes containing an anticoagulant are commerciallymanufactured and sold. An example of such a tube is disclosed in U.S.Pat. No. 5,667,963 to Smith et al. These additives do not specificallyhalt apoptosis nor do anything to protect cell morphology or function.

The present invention, therefore, is directed to methods and devices forcollecting a biological sample for the purpose of preserving cellmorphology, viability and function. More particularly, the invention isdirected to collection containers and to a method of collecting abiological sample and immediately contacting the sample with astabilizing additive or combination of additives to inhibit endogenouscaspases for the subsequent analysis or in vitro culture of the cells inthe biological sample. The stabilizing agent or agents of the inventionis a suitable mixture that is able to inhibit, prevent or reduce theoccurrence of apoptosis and apoptotic events during storage or cultureof the biological sample.

Accordingly, a primary aspect of the present invention is to provide amethod and device for collecting a biological sample, directly from apatient or animal in the presence of a stabilizer or stabilizer mixturecapable of inhibiting endogenous caspases for analysis of morphology orfunction of whole cells, cell constituents, or tissue, or maintainingthe viability of same for in vitro culture of cells or tissue. Thestabilizing additive is present in an effective amount to stabilize thebiological sample and to inhibit endogenous caspases for analysis ofcells, cell constituents, tissue or culture. Desirably, the sample iswhole blood or a tissue sample.

One aspect of the present invention is to prepare a biological samplethat is stable at room temperature for extended periods of time withlittle or no degradation in cell morphology, function, or composition.Accordingly, a method is provided for producing a biological sample thatis stable at room temperature for extended periods of time with littleor no incidence of morphological changes, cell membrane degradation, DNAfragmentation, or loss of cell function or viability.

A further aspect of the invention is to provide a method and device forinhibiting or eliminating incidence of morphological changes, cellmembrane degradation, DNA fragmentation, or loss of cell function orviability.

Another aspect of the invention is to provide a collection container forreceiving and collecting a biological sample where the container ispre-charged with a measured quantity of a stabilizing agent or mixtureof agents. The stabilizing agent may be supplied in the form of aliquid, a liquid or solid aerosol, a pellet, a powder or a gel to anysurface of the container.

A further aspect of the present invention is to provide a method forstabilizing a biological sample, particularly whole blood or a componentthereof, immediately upon collection from the patient to inhibit orprevent sample degradation when the sample is stored at varioustemperatures.

Another aspect of the present invention is to provide an evacuatedcontainer that is supplied with an effective amount of a stabilizingagent, where the container has an internal pressure sufficiently low todraw a predetermined volume of a biological sample into the container.

Still another aspect of the present invention is to provide a bloodcollection container for collecting an amount of blood and mixing theblood with a stabilizing agent or mixture of agents at the point ofcollection to produce a blood sample that is stable by preventingdegradation of the sample such that analysis of cell morphology,function or in vitro culture of the sample can be conducted at a latertime.

The aspects of the invention are attained by providing an apparatus forcollecting a biological sample. The apparatus generally includes acontainer comprising at least one interior wall that defines a reservoirportion for containing a volume of a biological sample and at least oneopening in communication with the reservoir portion. The containerincludes at least one stabilizing agent in an effective amount topreserve the biological sample and prevent or inhibit or eliminateincidence of morphological changes, cell membrane degradation, DNAfragmentation, or loss of cell function or viability. Preferably, thecontainer is pre-treated with the stabilizing agent prior to collectionof the sample.

The aspects of the invention are further attained by providing a methodof preparing a stable biological sample comprising providing a samplecollection container. Desirably, the container has a side wall and abottom defining an internal chamber where the interior of the containercontains at least one stabilizing agent in an amount sufficient toprevent or eliminate incidence of morphological changes, cell membranedegradation, DNA fragmentation, or loss of cell function or viability.The container may have attributes associated with promotion of in vitrocell culture. A biological sample is obtained and immediately introducedinto the container, and the biological sample is mixed with thestabilizing agent to form a stabilized biological sample.

The aspects of the invention are also attained by providing a method ofcollecting and stabilizing a whole blood sample. The method comprisesproviding a sample collection container having a side wall and a bottomforming an internal chamber. The container is provided with an effectiveamount of a stabilizing agent to stabilize cells in the whole bloodsample. The internal chamber has pressure less than atmosphericpressure. A whole blood sample is collected directly from a patient inthe collection container, and the blood sample is mixed with thestabilizing agent to form a stable whole blood sample. As the biologicalsample is drawn into the collection device, it is immediately exposed tothe stabilizing agent, and the process of protecting cell morphology,membrane integrity and function would begin immediately uponintroduction of the sample.

The aspects of the invention are also attained by providing a method ofcollecting and stabilizing a tissue or bone marrow or body fluidaspirate sample. The method comprises providing a sample collectioncontainer having a side wall and a bottom forming an internal chamber.The container is provided with an effective amount of a stabilizingagent or mixture of agents to stabilize cells in tissue or aspiratesample. A tissue or bone marrow or body fluid aspirate sample iscollected directly from a patient in the collection container, and thesample is mixed with the stabilizing agent to form a stable biologicalsample. As the biological sample is introduced into the collectiondevice, it is immediately exposed to the stabilizing agent, and theprocess of protecting cell morphology, membrane integrity and functionwould begin immediately upon introduction of the sample. The containermay also be provided with attributes that promote the culture or growthof cells. These attributes may include but not be limited to surfacecharge of interior surfaces of the container, porous membranes, cellnutrient media, or artificial scaffolding structures.

The method and collection device of the present invention have severaldistinct advantages. One advantage of the collection device is theoffering of a system, preferably a closed system, that includes thestabilizing agent and which protects the sample from deleteriousexposures. Still another advantage is routine line production of suchcollection devices, whereby quality control measures and procedures areapplied to the product. Yet another advantage is the standardization ofsuch collection devices where no industry standards currently exist.Moreover, the relevance of cell or tissue research and analysis isincreased by preserving and being able to characterize and study cellsin a state that is as close to the in vivo state as possible.

These aspects, advantages and other salient features of the presentinvention will become more apparent from the following detaileddescription of the invention, particularly when considered inconjunction with the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a typical blood collection tube.

FIG. 2 is a perspective view of a tissue culture vessel.

FIGS. 3A and 3B show a sample collection assembly.

FIG. 4 is a longitudinal sectional view of a syringe.

FIG. 5 is a longitudinal sectional view of another embodiment of asyringe.

FIG. 6 is a perspective view illustrating a blood collecting bag.

DETAILED DESCRIPTION OF THE INVENTION

While this invention is satisfied by embodiments in many differentforms, there will herein be described in detail preferred embodiments ofthe invention, with the understanding that the present disclosure is tobe considered as exemplary of the principles of the invention and is notintended to limit the invention to the embodiments illustrated anddescribed. Numerous variations may be made by persons skilled in the artwithout departure from the spirit of the invention. The scope of theinvention will be measured by the appended claims and their equivalents.

The present invention is directed to a method and device for stabilizinga biological sample to better enable analysis and in vitro culture ofcells and tissues. More particularly, the present invention is directedto a method and device for inhibiting apoptosis in a biological sampleduring storage or culture. According to the present invention, thedevice comprises a container containing an amount of a stabilizing agentfor admixing with a biological sample immediately on collection of thesample. Also according to the present invention, the method comprisesproviding a sample collection container containing a stabilizing agentin an amount sufficient to inhibit apoptosis and adding to the containera biological sample.

The biological sample is any body fluid or tissue sample withdrawn froma patient. Typically, the biological sample is whole blood or acomponent thereof, including umbilical cord blood or placental blood.Examples of other biological samples include cell-containingcompositions such as red blood cell concentrates, platelet concentrates,leukocyte concentrates, urine, bone marrow apirates, cerebral spinalfluid, tissue, fine needle organ or lesion aspirates, feces, saliva andoral secretions, nasal secretions lymphatic fluid and the like.

The sample collection system of the present invention can encompass anycollection device including, but not limited to, tubes such as testtubes and centrifuge tubes; closed system blood collection devices, suchas collection bags; syringes, especially pre-filled syringes; catheters;microtiter and other multi-well plates; arrays; tubing; laboratoryvessels such as flasks, spinner flask, roller bottles, vials, microscopeslides, microscope slide assemblies, coverslips, films and poroussubstrates and assemblies; pipettes and pipette tips, etc.; and othercontainers suitable for holding a biological sample. The interior of thevessel may be treated with the stabilizing agent.

Plastic or glass is often used to manufacture the collection device usedin the present invention. Some preferred materials used to manufacturethe collection device include polypropylene, polyethylene,polyethyleneterephthalate, polystyrene, polycarbonate and cellulosics.More expensive plastics such as polytetrafluoroethylene and otherfluorinated polymers may also be used. In addition to the materialsmentioned above, examples of other suitable materials for the collectiondevices used in the present invention include polyolefins, polyamides,polyesters, silicones, polyurethanes, epoxies, acrylics, polyacrylates,polysulfones, polymethacrylates, PEEK, polyimide and fluoropolymers suchas PTFE Teflon®, FEP Teflon®, Tefzel®, poly(vinylidene fluoride), PVDFand perfluoroalkoxy resins. Glass products including silica glass arealso used to manufacture the collection devices. One exemplary glassproduct is PYREX® (available from Corning Glass, Corning, N.Y.). Ceramiccollection devices can be used according to embodiments of theinvention. Cellulosic products such as paper and reinforced papercontainers can also be used to form collection devices according to theinvention.

The stabilizing agent of the invention is a suitable agent that is ableto inhibit caspase activity and the resultant apoptotic events duringstorage of a biological sample. The agent stabilizes the biologicalsample, such as a blood sample, to produce a stable composition thatinhibits or prevents apoptosis present in the biological sample. Inaccordance with the present invention, the collection device ispre-treated with the stabilizing agent, preferably by the manufacturer,and is packaged in a ready-to-use form. Typically, the packagedcollection device is sterile and is also packaged in sterile packagingmaterials.

The present invention could be used by clinical laboratories,pharmaceutical companies, biotechnology companies, contract researchorganizations, university researchers, research hospitals and anyinstitution and individual who is interested in studying or analyzingcells or tissues. The present invention would enable researchers andlaboratorians to conveniently and readily protect cellular samples fordownstream analysis. The collection device according to the presentinvention would serve as a front-end sample collection device aidinganalytical and processing objectives including, both to add not limitedto the following: flow cytometry, multiplexed bead assays cell surfacemarker identification and analysis, routine hematology assays includingCBC and white blood cell differential, HLA typing, cord blood collectionand stem cell harvesting, bone marrow aspirate collection and analysis,culture of non-immortalized cells, preservation of platelet viability,and preservation of DNA integrity.

Preferably, the stabilizing agent comprises or consists of at least onecaspase inhibitor, for example a caspase inhibitor which inhibits any orall cysteinyl aspartic acid proteases (Caspases 1-14). Caspaseinhibitors are known in the art, as shown for example in U.S. Pat. Nos.6,153,591, 6,184,210, 6,045,990, 6,355,618, 6,197,750, 5,869,519,6,200,969, and 6,242,422, and U.S. patent application 2002/0045623, thedisclosures of which are hereby incorporated by reference. Thestabilizing agent may be in any suitable form including, but not limitedto, solution, suspension or other liquid, pellet, tablet, spray-dried,freeze-dried, powder, particle, gel, crystals or lyophilized form. Thecaspase inhibitor may be in combination with other additives, asdiscussed below. Because the half-life of many inhibitors may be short,the stabilizing agent is preferably introduced into the collectiondevice in such a form so as to optimize the shelf life of the inhibitor.Lyophilization appears to be particularly useful in that it providesgood stability and also allows subsequent sterilization, both of whichare key from a standpoint of automation and standardization.

The stabilizing agent may be located on any surface of the collectiondevice. The stabilizing agent may also be located on stoppers and sealsfor closing such devices or on mechanical, or other, inserts placedwithin such devices. Preferably, the caspase inhibitor or admixturethereof is located anywhere along at least one interior wall of thecollection device or anywhere within the reservoir portion. In addition,some inhibitors may exhibit light sensitivity. Thus, it may be desirableto protect the agent from light. For such inhibitors, use of an opaquetube, e.g., an amber-colored tube, would be advantageous. Alternatively,placing the agent into a capsule that protects it from light exposure,e.g., in powdered form, and then placing the capsule into the tube wouldalso address this issue. Capsulating the agent may also prevent otherundesirable interactions between the agent and other elements in thecontainer. Capsule materials that dissolve upon sample collection arewell known in the art.

The caspase inhibitor or admixture thereof may be applied to thecollection device by any number of methods. For example, the caspaseinhibitor or admixture thereof may be spray dried, loosely dispensed orlyophilized over the surface of the interior wall of the collectiondevice. Alternatively, the stabilizing agent, such as when in gel orliquid form, for example, may be positioned in the reservoir portion ofthe collection or culture device.

The quantity and location of the caspase inhibitor or admixture thereofare determined by several variables, including the mode of application,the specific caspase inhibitor or admixture thereof used, the internalvolume and internal pressure of the collection device, and the volumeand type of the biological sample drawn or otherwise introduced into thecontainer.

The concentration of the caspase inhibitor or admixture thereof issufficient to inhibit the caspases and to prevent sample degradation ascompared to an identical device without such inhibitor. In addition tothe stabilizing agent, the device of the present invention may alsocontain carrier media (e.g., water, alcohol, dimethylsulfoxide),stabilizing media (e.g., polyvinylpyrollidone, trehalose, mannitol,dextrose) or one or more other additives such for treating thebiological sample. Suitable additives include, but are not limited to,alcohols, aldehydes, ketones, organic acids, salts of organic acids,alkali metal salts of halides, organic chelating agents, fluorescentdyes, antibodies, binding agents, anticoagulants such as sodium citrate,heparin, potassium or sodium salts of EDTA and the like, densitygradient media and any other reagent or combination of reagents normallyused to treat biological samples for analysis. Preferably, the carrierand additives do not inhibit cell function or decrease cell viability.Where the caspase inhibitor or admixture thereof is in tablet form,pharmaceutical tablet disintegrating materials may be included, ifdesired.

The method of the present invention is performed by obtaining abiological sample and introducing the sample into the containercontaining the caspase inhibitor. In preferred embodiments, thebiological sample is withdrawn from the patient directly into thecollection container without any intervening process steps. It has beenfound that collecting the biological sample directly from the patient,such as when collecting a whole blood sample, and introducing the sampledirectly into the container containing the stabilizing agentsubstantially prevents apoptosis that otherwise occurs when the sampleis stored before combining it with the caspase inhibitor. The method ofthe present invention is useful both with open collection systems andwith closed collection systems wherein the opening is closed by aclosure means.

In one embodiment, the collection device of the present invention is fordrawing a whole blood sample directly from a patient for inhibitingapoptosis immediately at the point of collection. The device may be anevacuated, a partially-evacuated or a non-evacuated system forcollecting blood. A suitable example of an evacuated system is a closedtube. A manual syringe draw is a suitable example of both apartially-evacuated and a non-evacuated system. Non-evacuated systemscan also include automatic draw systems. Evacuated systems areparticularly preferred.

Referring to the drawings in which like reference characters refer tolike parts throughout the several views thereof, FIG. 1 shows a typicalblood collection device 10, which includes a container 12 defining achamber 14. In the embodiment illustrated, container 12 is a hollow tubehaving a side wall 16, a closed bottom end 18 and an open top end 20.Optionally, a separating member 13 is provided within the containerchamber 14. Separating member 13 serves to assist in separatingcomponents of the sample, for example, by centrifugation. Container 12is dimensioned for collecting a suitable volume of biological fluid,preferably blood. A closure means 22 for covering open end 20 to closecontainer 12 is necessary where a sterile product is demanded. Forconventional tubes, a screw cap is normally sufficient. For evacuatedcollection tubes, a tight-fitting, elastomeric plug is generallyemployed to contain the vacuum during the required storage periods.Preferably, closure 22 forms a seal capable of effectively closingcontainer 12 and retaining a biological sample in chamber 14. Closure 22may be one of a variety of forms including, but not limited to, rubberclosures, metallic seals, metal-banded rubber seals and seals ofdifferent polymers and designs. A protective shield 24 may overlieclosure 22. Container 12 also contains a stabilizing agent in accordancewith the present invention.

Container 12 can be made of glass, plastic or other suitable materials.Preferably, container 12 is transparent. Non-limiting examples ofsuitable transparent thermoplastic materials for container 12 arepolycarbonates, polyethylene, polypropylene andpolyethyleneterephthalate. Plastic materials can be oxygen impermeablematerials or contain an oxygen impermeable or semi-permeable layer.Alternatively, container 12 can be made of a water and air permeableplastic material. The caspase inhibitor or admixture thereof may beprovided to the container using any appropriate means. In one aspect,the caspase inhibitor is in a liquid solution and is placed into thecontainer. Subsequently, the solution may be lyophilized by methods thatare known in the art, such as, for example, freeze drying. For example,by freezing the solution and then slowly warming after freezing, whilesimultaneously applying a vacuum, a freeze-dried powder remains in thecollection tube. An additive such as an excipient, for example, PVP ortrehalose, may also be added to the caspase inhibitor solution prior tofreeze drying so that the resulting stabilizing agent is pelletized inthe container. Vacuum drying may also be used after adding thestabilizing solution. In another aspect, the caspase inhibitor oradmixture thereof is formed into a liquid or solid aerosol and sprayedonto one or more surfaces of the interior of the container.

The pressure in chamber 14 is selected to draw a predetermined volume ofbiological sample into chamber 14. Preferably, closure 22 is made of aresilient material that is capable of maintaining the internal pressuredifferential between atmospheric pressure and a pressure less thanatmospheric. Closure 22 is such that it can be pierced by a needle 26 orother cannula to introduce a biological sample into container 12 asknown in the art. Preferably, closure 22 is resealable. Suitablematerials for closure 22 include, for example, silicone rubber, naturalrubber, styrene butadiene rubber, ethylene-propylene copolymers andpolychloroprene.

Suitable examples of container 12 include single-wall and multi-layertubes. A more specific example of a suitable container 12 is disclosedin U.S. Pat. No. 5,860,937 to Cohen, which is hereby incorporated byreference in its entirety.

As noted, container 12 may also contain a gel mechanical or otherseparating member (e.g., filter paper or the like). In such cases, thestabilizing agent may be spray dried and/or lyophilized on an exteriorsurface of the separation media. Container 12 may also be a collectiondevice for blood plasma preparation. Such a collection device comprises,in addition to the stabilizing agent, an element for separating plasmafrom human or animal whole blood. The element for separating plasma fromwhole blood may be a separating member such as a gel formulation or amechanical media. The gel is desirably a thixotropic polymeric gelformulation. The gel may be a homopolymer or a copolymer and may includesilicone-based gels such as, for example, polysiloxanes, or organichydrocarbon-based gels such as, for example, polyacrylics, polyesters,polyolefins, oxidized cis polybutadienes, polybutenes, blends ofepoxidized soybean oil and chlorinated hydrocarbons, copolymers ofdiacids and propandiols, hydrogenated cyclopentadienes and copolymers ofalpha-olefins with dialkylmaleates. The gel desirably isolates theplasma from the cells of the blood sample in the tube by serving as adensity separation medium. An example of a suitable plasma preparationtube is disclosed in U.S. Pat. No. 5,906,744 to Carroll et al., which ishereby incorporated by reference in its entirety. In this way,stabilization can be provided both before, during and aftercentrifugation to separate the plasma from the blood. In the case of agel separating material, it may be desirable to providephysical/chemical separation between the stabilizing agent and the gel,e.g., use of a capsule as discussed above. For example, if portions ofthe agent are incorporated into or react with the gel, the effectivenessof the agent may be reduced. For the same reasons, where a mechanicalseparating element is used, the element is desirable substantially inertto the stabilizing agent, and this reflects a significant advantage ofsuch a separator. Providing a separating element in plasma tubes, versuscentrifuging without a separating element, is particularly advantageous.Specifically, because cell lysing may release proteases that induceapoptosis, the better the separation between the cells (i.e., theclotted blood) and the plasma, the better the stability of proteins inthe plasma sample. Useful mechanical separators are found, for example,in U.S. Pat. Nos. 6,516,953; 6,406,671; 6,409,528; and 6,497,325, thecontents of which are hereby incorporated by reference in theirentirety.

Container 12 may also be collection tube for centerifugally separatinglymphocytes and monocytes from heavier phases of a sample of whole bloodcomprising, in addition to the stabilizing agent, a liquid densitygradient medium and a means for preventing mixing of the liquid densitygradient medium with a blood sample prior to centrifugation. An exampleof a suitable lymphocyte/monocyte collection tube is disclosed in U.S.Pat. No. 5,053,134 to Luderer et al., which is hereby incorporated byreference in its entirety.

Other commercially available blood collection tubes suitable for use inthe present invention include the following, all of which are sold byBecton Dickinson and Company, Franklin Lakes, N.J., with allregistrations and trademarks belonging to Becton Dickinson and Company:VACUTAINER® hematology tubes, catalog nos. 367650-1, 367661, 6405, 6385,6564, 367653, 367665, 367658, 367669, 6450-8, 6535-37 and 367662;VACUTAINER® K₂EDTA tubes, catalog nos. 367841-2, 367856 and 367861;VACUTAINER® PST tubes, catalog nos. 367793-4, 6698, 6595 and 6672;VACUTAINER® CPT tubes, catalog nos. 362753 and 362760-1; VACUTAINER® SSTtubes, catalog nos. 367782-89, 6509-17 and 6590-92; and VACUTAINER® ACDtubes catalog nos. 367756, 364012 and 4816.

In another embodiment, the invention provides a kit having at least twocontainers comprising one or more stabilizing agents. For example, thekit may comprise a primary collection tube, e.g., a plasma separatingtube having a separating element therein, and a secondary tube fortesting, e.g., for pouring or otherwise dispensing the collected plasmainto. Both would have stabilizing agent(s) therein. Optionally, the kitcould include a tube-to-tube transfer device to prevent the need forpouring or other unsafe transfer practices, in which case the secondarytube would be at a reduced pressure to draw in the plasma One using sucha kit would collect a sample in the primary tube, centrifuge, transferthe sample of interest to the secondary testing tube, and perform thetesting. The secondary testing could be of a variety of sizes, dependingon the desired testing.

In an embodiment, the container is a tube with two open ends havingclosures thereon. Such a tube would allow one to sample, e.g., for aplasma separating tube with a separating element therein, either theplasma sample or the clot sample.

In another embodiment, the collection device of the present inventioncomprises a tissue culture vessel such as, for example, a single- ormulti-well plate, a microtiter plate, a tissue culture plate or flask orthe like. A typical test plate generally comprises one or more wells,which are preferably cylindrical. As shown in FIG. 2, a test plate 30includes an upper surface 32 and a lower surface 34. Test plate 30further includes a number of wells 36 each comprising a sidewall 38extending from upper surface 32 of the plate to lower surface 34 of theplate. Each well comprises a top portion 40 and a bottom portion 44. Topportion 40 comprises an open end 42, that extends to bottom portion 44that comprises a closed end 46. Bottom portion 44 may be flat, conical(pointed) or rounded. The capacity of each well 36 typically ranges fromseveral milliliters (ml) to less than about 0.5 ml. Wells 36 may eachaccommodate therein a stabilizing agent according to the presentinvention.

The number of wells 36 in test plate 30 is not critical. There may beany number of wells, although six, twelve, twenty-four, forty-eight andninety-six well test plates are commonly known and available. In FIG. 2,a six-well test plate is illustrated, merely for exemplary purposes, andthe invention is not dependent upon the number of wells. Most standardmulti-well plates have the wells arranged in orthogonal rows and columnsso as to be able to clearly identify the individual wells being used. Ofcourse, the arrangement of the wells in test plate 30 is not anessential limitation of the present invention because any arrangement ofwells is contemplated by the invention.

Plate 30 may be formed from thermoplastic materials by vacuum forming,sheet molding, injection molding or other similar techniques. Suitablethermoplastic materials include, but are not limited to, polystyrene,polyvinylchloride, polycarbonate, polyethyleneterephthalate and thelike. Preferably, plate 30 is transparent.

Surrounding the wells and forming the outside border of test plate 30are sidewalls 38. In the present embodiment, test plate 30 has six (6)sidewalls. Well known test plates are rectangle or quadrilaterallyshaped, although for purposes of the present invention the plate may befabricated in any practical configuration. Examples of suitable testplates containing a plurality of wells are disclosed in U.S. Pat. No.5,882,922 to Tyndorf et al., U.S. Pat. No. 5,801,055 to Henderson andU.S. Pat. No. 5,681,743 to Brian et al., each of which is herebyincorporated by reference in its entirety.

In yet another embodiment, the collection device according to thepresent invention may be a sample collection assembly for thecollection, transport and dispensing of biological samples. Thecollection assembly generally includes a plurality of sample wells forcollecting individual biological samples. The sample wells are supportedin a sample tray in a spaced-apart orientation. The sample tray may besupported within a case that encloses the sample tray and allows thesafe and efficient transport of the sample wells. The sample tray ismovably accommodated within the case for movement between a firstposition enclosing the plurality of sample wells, to a second positionrendering exteriorly accessible one of the sample wells so that thesample can be manually dispensed from the tray.

As shown in FIGS. 3 a and 3 b, sample tray 50 includes a plurality oflongitudinally spaced depressions forming specimen collection wells 52.Sample tray 50 may be formed of a suitably deformable plastic material.Wells 52 have a bottom 54 and an open end 56. It is contemplated thatthe sample wells may be in the shape of open ended cup-like members.Wells 52 are constructed to have sufficient depth so as to retain asuitable volume of a biological sample. Wells 52 may each accommodatetherein a stabilizing agent according to the present invention. Whiletray 50 of the present invention is shown having a single row of wells52 formed therein, the present invention contemplates that the wells maybe provided in any number or any array desirable for a particulartesting situation. The sample collection assembly may include a samplecollection case 57. Upon collection of a biological sample within wells52, sample tray 50 may be inserted into the open end 58 of samplecollection case 57 and then within the interior 59 of sample collectioncase 57 until all of wells 52 are enclosed therein. A suitable samplecollection assembly is disclosed in U.S. Pat. No. 6,357,583 B1 toRainen, which is hereby incorporated by reference in its entirety.

According to another embodiment of the present invention, the collectiondevice comprises a syringe and, more preferably, a pre-filled syringe. Atypical syringe comprises a generally cylindrical barrel having opposedproximal and distal ends with at least one chamber formed between theends for receiving a substance such as a biological sample. A plunger istypically sealably disposed within the barrel and movable with respectthereto, and sealing means may be sealably disposed approximate to thedistal end of the barrel. Referring now to FIG. 4, there is shown asyringe 60, which includes an elongate barrel or cylinder 62 having anopen, proximal end 64 and a distal end 66, with at least one hollowchamber 68 formed between the proximal and distal ends for receiving abiological sample. In the embodiment illustrated, distal end 66 includesa needle guard 70. The needle guard keeps the syringe, as well as theneedle, sterile during storage.

The barrel of the syringe includes a stabilizing agent. Preferably, thebarrel of the syringe is pre-filled with the stabilizing agent.Pre-filled syringes, as the term is known in the art, are syringes thatare filled by the manufacturer and shipped to the health care providerready for use.

A plunger 72 may be situated at open, proximal end 64. Plunger 72 can bemoved by means of a plunger rod 74, which is secured to the plunger, forexample, by screwing. At the same end where the plunger is situated, thebarrel may have a fingergrip 76, which is secured to the barrelaccording to the so-called snap-cap principle. Fingergrip 76 preferablyconsists of slightly resilient material, for example plastics. Inanother embodiment (not shown), the fingergrip is a flange-like part ofthe barrel projecting radially outwards. Of course, other constructionsknown to those skilled in the art are possible.

A stopper 78, which closes the barrel, may be situated in the end of thebarrel remote from the plunger. The plunger and the stopper arepreferably manufactured from an elastic material and, most preferably,from rubber of a pharmaceutical quality.

In the embodiment illustrated, an injection needle 80 is secured to thebarrel by means of a needle holder 82. The needle holder has a neck 84,which holds the needle, a shaft 86 and a collar 88. The needle holder ispreferably manufactured from slightly resilient material that hasresistance to deformation such as, for example, plastics, and is securedto the end of the barrel by means of a snap-cap construction. In thealternative, the needle holder may be secured to the barrel by means ofa screwed or adhesive connection or, when the barrel also comprises acollar, by means of a clamping ring; in the latter embodiment, theneedle holder may also be flanged around a collar of the barrel.

Although the syringe barrel illustrated in this embodiment includes alocking Luer-type collar 88, it is within the purview of the presentinvention to include syringe barrels without a collar, syringe barrelshaving an eccentrically positioned nozzle, and various other nozzle-likestructures adapted to accept, either permanently or removably, a needlecannula or needle cannula assembly. It is only required that there is anaperture on the distal end of the syringe barrel in fluid communicationwith the interior of the syringe barrel.

One or more slots 90 may be recessed in the inner wall of shaft 86 andthe rear face of neck 84. The slot or slots extend into the rear end ofthe cannula. In cross-section, the slots may be parts of a circle, butother shapes are also possible, provided the size is such thatsufficient injection liquid can be readily passed through; this isachieved if the diameter of the slot or the overall cross-section of theslots is at least as large as that of the cannula. Shaft 86 of needleholder 82 is constructed so that when stopper 78 slides axially forward,it is received, with friction, by the shaft; therefore, apart from slots90 recessed in the shaft, the inside diameter of the shaft isapproximately as large as that of barrel 62. Shaft 86 of needle holder82 is slightly longer than stopper 78 so that the part 92 of the slot(s)adjoining the barrel is free when the stopper is moved forward againstthe rear wall of the neck of the needle holder. If desired, needle guard70 may be constructed to also serve as a plunger rod. In that case,prior to use of the syringe, the needle guard is removed from the needleand secured at the other end of the syringe to the plunger.

Generally, a syringe comprising a needle protector has a safety member,which indicates whether the needle protector has previously beenremoved. Such a safety member in the form of a cap is described, forexample, U.S. Pat. No. 3,995,630.

In further embodiments, the syringe is not stored with a needle inposition, i.e., it is a needleless syringe as known in the art. This isillustrated in FIG. 5. With such a syringe, before use, the needle ispositioned on neck 84 of needle holder 82 by means of a needle hub. Aso-called Luer cone is preferably used for this connection. In thisembodiment, aperture 94 in the neck of the needle holder is closed onthe outside by a protective cap 96, which ensures the sterility of thesyringe as well as the needle holder. Slot 90 recessed in the needleholder projects into the end of the neck aperture.

An example of a suitable syringe is disclosed in U.S. Pat. No. 6,027,481to Barrelle et al., which is hereby incorporated by reference in itsentirety. Other examples of suitable syringes are disclosed in, forexample, U.S. Pat. No. 4,964,866 to Szwarc, U.S. Pat. No. 4,986,818 toImbert et al., U.S. Pat. No. 5,607,400 to Thibault et al. and U.S. Pat.No. 6,263,641 B1 to Odell et al., each of which is hereby incorporatedby reference in its entirety.

The collection device of the present invention may also comprise acollection bag suitable for holding a biological sample such as, forexample, a blood collecting bag, a blood plasma bag, a buffy coat bag, aplatelet bag or the like. For ease of description, a blood collectingbag will now be described with reference to FIG. 6.

FIG. 6 illustrates a blood collecting bag 300 for accommodatingcollected blood. Blood collecting bag 300 has a body 302 formed bysuperposing a pair of identically cut pieces of a sheet material made ofa resin, which will be more specifically described hereinafter, andpossessed of flexibility and fusing (i.e., heat fusion, high frequencyfusion or the like) or adhesively joining to each other the periphery ofthe sealing portion 304 of each of the pieces of sheet material. Ablood-accommodating portion 306 accommodating collected blood is formedat an inner portion surrounded with sealing portion 304 of body 302.Blood collecting bag 300 preferably contains a stabilizing agent inaccordance with the present invention.

One end of the flexible tube 308 communicating with blood-accommodatingportion 306 is connected with body 302 at an upper portion thereof. Ablood collecting needle 310 is installed at the other end of flexibletube 308 through a hub 312. A cap 314, which is to cover bloodcollecting needle 310, may be installed on hub 312. Two openings 316 and318, each sealed with a peel tab, may be formed at an upper portion ofbody 302 such that they can be opened.

The composition, characteristics and the like of the material of thesheets composing body 302 of blood collecting bag 300 are not limited tospecified ones. In this case, as the sheet material composing bloodcollecting bag 300, soft polyvinyl chloride or materials containing thesoft polyvinyl chloride as their main component is preferably used. Forexample, a copolymer containing the soft polyvinyl chloride as its maincomponent and a small amount of macromolecular material, a polymerblend, a polymer alloy and the like can be used. As the plasticizer forthe soft polyvinyl chloride, dioctylphthalate (DEBHP,di(2-ethylhexyl)phthaldte) and (DnDP, di(n-decyl)phthalate) can bepreferably used. The content of such a plasticizer in the polyvinylchloride is preferable to be in the approximate range of 30 to 70 partsby weight, based on 100 parts by weight of polyvinyl chloride.

The other substances which are effectively usable for the sheet materialof blood collection bag 300 are polyolefins, ie., the products ofhomopolymerization or copolymerization of such olefins or diolefins asethylene, propylene, butadiene, and isoprene. As typical examples,polyethylene, polypropylene, ethylene vinyl acetate copolymer (EVA),polymer blends formed between EVA and various thermoplastic elastomers,and arbitrary combinations thereof may be cited. Besides, suchpolyesters as polyethylene terephthalate (PET), polybutyleneterephthalate (PBT), poly-1,4-cyclohexane dimethyl terephthalate (PCHT)and polyvinylidene chloride are also usable.

In yet another embodiment, the collection device of the presentinvention may be a laboratory vessel that contains caspase inhibitor oradmixture thereof. Particular vessels that can be used in accordancewith the present invention include, for example, vials, flasks, spinnerflasks, roller bottles, microscope slides, microscope slide assemblies,sample chambers for analytical devices, tapes, laminates, arrays,catheters, pipettes, tubing and the like. Laboratory vessels accordingto the present invention have at least one operational surface. Manyvessels according to the invention have at least one interior wall,which defines a reservoir portion for containing the biological sample,and at least one opening in communication with the reservoir portion.

Plastic or glass is often used to manufacture the laboratory vessels.Some preferred materials used to manufacture laboratory vessels includepolypropylene, polyethylene, polyethyleneterephthalate, polystyrene,polycarbonate and cellulosics. Because polypropylene is inexpensive, itis a particularly preferred material for laboratory vessels used forhandling and transporting minute and precise amounts of biologicalsample.

Examples of other suitable materials for the laboratory vessels of thepresent invention include polyolefins, polyamides, polyesters,silicones, polyurethanes, epoxies, acrylics, polyacrylates, polyesters,polysulfones, polymethacrylates, PEEK, polyimide and fluoropolymers.Glass products including silica glass are also used to manufacturelaboratory vessels.

In another embodiment, cells stabilized by the articles and/or processesof the inventions are used as therapies. For example, cells fromumbilical cord blood have been used as therapy, e.g., by transplantingsub cells to patients having genetic or blood disorders. Stem cellsfound in the cord bloods are believed to replace or supplementnon-functioning or malfunctioning cells of the recipient. It is alsobelieved that such stem cells may help regenerate damaged tissues.

According to the invention, umbilical cord blood (or placental blood) isstabilized upon collection by use of a container comprising one or morecaspase inhibitors, as discussed above. The blood is then transplantedto a human being, either after being cryopreserved, or fresh.(Cryopreservation techniques are know to those skilled in the art.)

Similary, it is possible to collect a product of leukapheresis in acontainer comprising one or more caspase inhibitors, and administeringthat product to a patient, again either after being cryopreserved orfresh. (Leukapheresis, as know in the art, is a process in which bloodis drawn, a specific cell product is separated out, and the remainder ofthe blood is returned to the subject.

By using a collection container comprising one or caspase inhibitor, thestability of stem cells would be expected to improve, relative to anidentical container without such caspase inhibitors. As a result, thetherapeutic effect of the stabilized product would be expected toimprove, as well. Advantagously, the caspase inhibitors do notdetrimentally effect cryopreservation steps, and are substantiallybenign to the human body upon injection.

1. An apparatus for containing a biological sample, comprising: acontainer having a reservoir portion for receiving the sample; and astabilizing agent in the reservoir of the container, the agentcomprising a caspase inhibitor.
 2. The apparatus of claim 1, wherein thecontainer is selected from the group consisting of tubes, closed systemblood collection devices, collection bags, syringes, pre-filledsyringes, catheters, microtiter plates, multi-well collection devices,flasks, spinner flasks, roller bottles, vials, pipettes, pipette tipsand tissue and other biological sample collection containers.
 3. Theapparatus of claim 1, wherein the container is a tube having a first endand a second end.
 4. The apparatus of claim 3, further comprising aseparating member disposed in the container.
 5. The apparatus of claim4, wherein the separating member is a mechanical separating element. 6.The apparatus of claim 5, wherein the mechanical separating element isat least partially coated with the at least one stabilizing agent. 7.The apparatus of claim 5, wherein the mechanical separating element issubstantially inert with respect to the stabilizing agent.
 8. Theapparatus of claim 4, wherein the separating member is a gel.
 9. Theapparatus of claim 8, wherein the gel separating member is physicallyseparated from the stabilizing agent.
 10. The apparatus of claim 1,wherein the stabilizing agent is in a form selected from the groupconsisting of a solution, suspension or other liquid, a pellet, atablet, a capsule, a spray-dried material, a freeze-dried material, apowder, a particle, a gel, crystals or a lyophilized material.
 11. Theapparatus of claim 10, wherein the stabilizing agent is lyophilized. 12.The apparatus of claim 1, wherein the caspase inhibitor inhibits one ormore cysteinyl aspartic acid proteases.
 13. The apparatus of claim 1,wherein the stabilizing agent comprises more than two caspaseinhibitors.
 14. The apparatus of claim 1, further comprising a carriermedia.
 15. The apparatus of claim 1, further comprising a stabilizingmedia.
 16. The apparatus of claim 15 wherein the stabilizing media istrehalose.
 17. The apparatus of claim 1, further comprising at least oneantioxidant.
 18. The apparatus of claim 1, further comprising at leastone reducing agent.
 19. The apparatus of claim 1, further comprising atleast one buffering agent.
 20. The apparatus of claim 3, furthercomprising a closure means for sealing the first end.
 21. The apparatusof claim 20, wherein the tube is partially evacuated.
 22. The apparatusof claim 21, wherein the stabilizing agent is lyophilized.
 23. Theapparatus of claim 22, wherein the stabilizing agent comprises more thantwo caspase inhibitors.
 24. The apparatus of claim 23, wherein the tubefurther comprises an anticoagulant.
 25. The apparatus of claim 24,wherein the anticoagulant is spray-dried onto at least a portion of aninterior wall.
 26. The apparatus of claim 25, wherein the anticoagulantcomprises a salt of EDTA.
 27. The apparatus of claim 24, wherein theanticoagulant comprises heparin.
 28. A tube for collecting andstabilizing a biological sample, comprising: a first end, a second endand at least one interior wall defining a reservoir portion forreceiving the sample; at least one stabilizing agent in the reservoir ofthe container, the stabilizing agent comprising a caspase inhibitor; athixotropic polymeric gel in the reservoir; and an element formaintaining separation of the stabilizing agent and the gel.
 29. Thetube of claim 28, wherein the element for maintaining separation is acapsule.
 30. The tube of claim 29, further comprising a closure meansfor sealing the first end.
 31. The tube of claim 30, wherein the closuremeans is pierceable by a needle for supplying the sample to the tube.32. The tube of claim 30, wherein the tube is partially evacuated. 33.The tube of claim 32, wherein the stabilizing agent is lyophilized. 34.The tube of claim 33, wherein the stabilizing agent comprises more thantwo caspase inhibitors.
 35. The tube of claim 33, wherein the tubefurther comprises an anticoagulant spray-dried onto at least a portionof the interior wall.
 36. A tube for collecting and stabilizing abiological sample, comprising: a first end, a second end and at leastone interior wall defining a reservoir portion for receiving the sample;at least one stabilizing agent in the reservoir of the tube the agentcomprising a caspase inhibitor; and a mechanical separating element inthe reservoir.
 37. The tube of claim 36, wherein the mechanicalseparating element is substantially inert with respect to thestabilizing agent.
 38. The tube of claim 36, further comprising aclosure means for sealing the first end.
 39. The tube of claim 38,wherein the closure means is pierceable by a needle for supplying thesample to the tube.
 40. The tube of claim 38, wherein the tube ispartially evacuated.
 41. The tube of claim 40, wherein the stabilizingagent is lyophilized.
 42. The tube of claim 41, wherein the stabilizingagent comprises more than two caspase inhibitors.
 43. The tube of claim41, wherein the tube further comprises an anticoagulant spray-dried ontoat least a portion of the interior wall.
 44. A kit for collecting andstoring a biological sample for subsequent testing, comprising: aprimary collection tube having a separator element therein; and asecondary tube; wherein the primary collection tube and the secondarytube contain one or more stabilizing agents, the agents comprising oneor more caspase inhibitors.
 45. The kit of claim 44, wherein theseparator element is a mechanical separating element.
 46. The kit ofclaim 45, wherein the mechanical separating element is at leastpartially coated with the one or more stabilizing agents.
 47. The kit ofclaim 46, wherein the mechanical separating element is substantiallyinert with respect to the one or more stabilizing agents.
 48. The kit ofclaim 44, wherein the separator element is a gel, and the gel isphysically separated from the stabilizing agent.
 49. The kit of claim44, further comprising a tube-to-tube transfer device.
 50. The kit ofclaim 49, wherein the second tube is maintained at a pressure to drawthe sample from the first tube through the tube-to-tube transfer deviceand into the second tube.
 51. A method of stabilizing a biologicalsample, comprising: providing a sample collection container; anddisposing the biological sample into the collection container such thatthe sample is contacted with a stabilizing agent comprising a caspaseinhibitor.
 52. The method of claim 51, wherein the sample collectioncontainer includes the stabilizing agent before collecting thebiological sample.
 53. The method of claim 51, wherein the disposing ofthe biological sample into the container and the contacting of thesample with the stabilizing agent are performed in the same collectioncontainer.
 54. The method of claim 53, wherein the collection containeris evacuated and has a predetermined internal pressure sufficient todraw a predetermined volume of the sample into the collection container.55. The method of claim 51, wherein the collection container is selectedfrom the group consisting of tubes, closed system blood collectiondevices, collection bags, syringes, microtiter plates, multi-wellcollection devices, flasks, spinner flasks, roller bottles and vials.56. The method of claim 51, wherein the stabilizing agent comprises morethan two caspase inhibitors.
 57. The method of claim 51, wherein thebiological sample is selected from the group consisting of whole bloodor a component thereof, umbilical cord or placental blood, red bloodcell concentrates, platelet concentrates, leukocyte concentrates,plasma, serum, urine, bone marrow apirates, cerebral spinal fluid,tissue, cells, feces, saliva and oral secretions, nasal secretions andlymphatic fluid.
 58. The method of claim 57, wherein the biologicalsample is whole blood.
 59. The method of claim 58, wherein the wholeblood is collected from a patient directly into the collectioncontainer.
 60. The method of claim 59, wherein the collection containerincludes the stabilizing agent before the blood is collected from thepatient.
 61. A method for making a collection container for collecting abiological sample, comprising: providing a collection container;disposing a stabilizing agent comprising at least one caspase inhibitorinto the container; lyophilizing the stabilizing agent; evacuating andsealing the container; and sterilizing the container.
 62. The method ofclaim 61, wherein the collection container is a tube.
 63. The method ofclaim 62, further comprising placing into the tube a separating member.64. The method of claim 63, wherein the separating member is amechanical separating element.
 65. The method of claim 63, wherein theseparating member is a gel.
 66. A method for treating, comprising thesteps of: collecting a cell population that comprises hematopoetic stemcells, wherein the cell population is collected in a containercomprising one or more caspase inhibitors; and administering at least aportion of the collected cell population into a patient.
 67. The methodof claim 66, wherein umbilical cord blood or placental blood iscollected into the container.
 68. The method of claim 66, wherein atleast a portion of the one or more caspase inhibitors are administeredinto the patient along with the at least a portion of the collected cellpopulation.
 69. The method of claim 66, further comprising the step ofcryopreserving the collected cell population, and thawing the collectedcell population, prior to the step of administering.
 70. The method ofclaim 66, wherein the container comprises at least two caspaseinhibitors.
 71. The method of claim 69, wherein at least a portion of acryopreservative used in the cryopreserving step is administered intothe patient along with the at least a portion of the one or more caspaseinhibitors and the at least a portion of the collected cell population.72. A method for treating, comprising the steps of: performing aleukapheresis process to collect a cell population that compriseshematopoetic stem cells, wherein the cell population is collected in acontainer comprising one or more caspase inhibitors; and administeringat least a portion of the collected cell population into a patient. 73.The method of claim 72, wherein at least a portion of the one or morecaspase inhibitors are administered into the patient along with the atleast a portion of the collected cell population.
 74. The method ofclaim 72, further comprising the step of cryopreserving the collectedcell population, and thawing the collected cell population, prior to thestep of administering.
 75. The method of claim 72, wherein the containercomprises at least two caspase inhibitors.
 76. The method of claim 74,wherein at least a portion of a cryopreservative using in thecryopreserving step in administered into the patient along with the atleast a portion of the one or more caspase inhibitors and the at least aportion of the collected cell population.